Adenylate kinases play a key role in the regulation of energy balance within cells, particularly maintenance of the ratio of ATP with its diphosphate (ADP) and monophosphate forms (AMP). ATP serves as the primary source of energy for biochemical reactions in cells and is also a key precursor in DNA and RNA synthesis during cellular growth and replication. The energy associated with the terminal phosphate bonds of ATP may be transferred to other nucleotides using a nucleoside monophosphate kinase such as adenylate kinase. In this manner, the terminal energy-rich phosphate bonds of ATP may be transferred to the appropriate nucleotides for use in a variety of biosynthetic and energy-requiring processes, such as biosynthesis of macromolecules, active ion transport, muscle contraction, thermogenesis, etc. A number of these energy-requiring biosynthetic reactions hydrolyze ATP into AMP plus pyrophosphate. Reutilization of the resulting AMP requires conversion back into the triphosphate form following conversion to ADP. Various nucleotide monophosphate kinases carry out the first step of phosphorylating AMP to its diphosphate form at the expense of ATP. In the case of adenylate kinase, this reversible reaction is given as AMP+ATP≡2 ADP.
Adenylate kinases also play a role in regulating the flow of carbon between net accumulation of glucose via the gluconeogenesis pathway and its subsequent catabolism via the glycolytic pathway by way of their control over the ratio of AMP to ATP. AMP is a positive allosteric effector of the enzyme 6-phophofructo-1-kinase, which shifts, and a negative allosteric effector for the enzyme fructose-1,6-bisphosphatase. When the first of these enzymes is activated, carbon flow is shifted in the direction of glycolysis; when the second of these enzymes is activated, carbon flow shifts in the direction of gluconeogenesis. Thus, increases in the ratio of AMP to ATP shift carbon flow toward glycolysis, while decreases in the ratio of AMP to ATP shift carbon flow toward glucose formation.
These enzymes have been studied in a number of mammals, including rat, porcine, chicken, bovine, rabbit, and humans. Evidence from biochemical studies suggests that human tissues have five adenylate kinase isozymes, AK1-AK5. Thus far the cDNAs of human AK1, AK2, AK4, and AK5 have been cloned. Adenylate kinase isoforms in humans are sequence related and also related to UMP/CMP kinases from several species. See Rompay et al. (1999) Eur. J. Biochem. 261:509-516, and the references cited therein.
The adenylate kinase isozymes AK1 (or myokinase), which is a cytosolic enzyme present in brain, skeletal muscle, and erythrocytes, and AK2, which is associated with the mitochondrial membrane in liver, spleen, heart, and kidney, both utilize ATP as their nucleoside triphosphate donor substrate. AK3 (or GTP:AMP phosphotransferase) is located in the mitochondrial matrix, primarily in heart and liver cells, and uses MgGTP instead of MgATP. AK4 and AK5 are both localized in brain tissue.
Several regions of AK family enzymes are well conserved, including the nucleoside triphosphate binding glycine-rich region, the nucleoside monophosphate binding site, and the lid domain that closes over the substrate upon binding (see Schulz (1987) Cold Spring Harbor Symp. Quant. Biol. 52:429-439).
These enzymes assist with maintenance of energy production and utilization within cells, particularly in cells having high rates of growth and metabolic activity such as in heart, skeletal muscle, and liver. In fact, adenylate kinase deficiency has been linked to hemolytic anemia and neurological disorders such as neurofibromatosis (Xu et al. (1992) Genomics 13:537-542. In addition, targeting regulation of ATP synthesis has been the basis of antiproliferative drugs for treatment of viral infections and cancer.
Adenylate kinases are also useful for activating nucleoside analogues used as pharmaceuticals, especially for cancer and viral infection. Most of these analogues must be phosphorylated to the triphosphate form in order to be pharmaceutically active. The first phosphorylation step in the activation of nucleoside analogs is catalyzed by deoxyribonucleoside kinases. Phosphorylation to the di- and triphosphates are then required.
Accordingly, adenylate kinases are a major target for drug action and development. Thus, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown adenylate kinases. The present invention advances the state of the art by providing a previously unidentified human adenylate kinase.